World globalization and the increased threat of bioterrorism have mandated rapid evaluation of immunization strategies for a panel of agents including Ebola (EBO), SARS and H5N1 viruses for which there are limited clinical or biological data. To date, the most effective Ebola immunization protocols have involved parenteral administration of a vesicular stomatitis virus (VSV)-based vector expressing the Ebola glycoprotein (EBOGP) or administration of recombinant adenovirus serotype 5 (Ad) expressing EBOGP. Although Ad vaccine carriers have a substantial amount of clinical data to support their use, they face the problem of widespread pre-existing immunity in the human population. To date, vaccination strategies against EBO involve use of Ad as the vaccine carrier but do not address this important issue. Studies proposed have three major aims employing Ad as a model vaccine carrier and EBOGP as a model antigen. We plan to test the hypothesis that mucosal administration (oral or nasal) can circumvent pre-existing immunity against Ad and confer immunity against Ebola challenge in several animal models. Mucosal immunization against EBO and other filoviruses has not been explored, yet immunization through the respiratory and gastrointestinal tract can effectively induce both local and systemic immune responses against many pathogens. Although there is some suggestive evidence that mucosal immunity plays a significant role in protection from EBO challenge, it is not clear what the influence of both the innate and adaptive immune response and the interaction between the two bears on the efficacy of EBO immunization strategies. Studies detailed in this proposal are specifically designed to fully characterize T and B cell mediated immune responses that develop systemically and locally after mucosal immunization before and after challenge with EBO in the presence and absence of pre-existing immunity to the Ad carrier. We also propose several formulation and biochemical strategies to promote and further strengthen the immune response against EBOGP during mucosal immunization. Results from these studies will further the understanding of EBO infection and identify specific elements of the immune response necessary to confer immunity to EBO. This information will be of value in the development of immunization strategies and identify targets for anti-viral regimens. They will also lead to development of effective and attractive vaccination strategies that can be applied to other pathogens. [unreadable] [unreadable] [unreadable] [unreadable]